FENESTRAE are circular transcellular pores in vascular endothelial cells playing critical roles in the maintenance of normal endothelial barrier function, blood homeostasis and ultimately survival. Fenestrae are spanned in most cases by a protein barrier called a fenestral diaphragm (FD). There is a significant knowledge gap in our understanding of the basic cell biological mechanism of fenestrae assembly, which is the subject of this proposal. Recently, our group demonstrated that Plasmalemma Vesicle Associated Protein (PLVAP or PV1) is critical for FD assembly, and that absence of PV1 in mice and humans causes abnormal fenestrae and early postnatal lethality due to multiple vascular defects. Critically for this proposal, PV1 is the only known marker for fenestrae, and we will exploit this property in our investigation of fenestrae assembly mechanisms. Our central hypothesis is that fenestrae assembly follows a three-step model: In Step 1, controlled actin depolymerization leads to cell thinning and apposition of apical and basal plasma membranes to within 50 nm. In Step 2, fenestrae pores form by fusion of exocytic vesicles with apical and basal membranes. In Step 3, the FD assembles using exocytosed PV1 and its interacting partners. This hypothesis is based on our key new observations including: 1) Arp2/3 complex inhibition induces fenestrae assembly; 2) de novo formation of the FD requires exocytosis of PV1 from an internal pool; 3) fenestrae assembly does not require caveolin 1, caveolae or PV1 endocytosis. We will critically test specific aspects of this model, using a functionally validated endothelial cell culture system. In Aim 1 we will define the role and mechanism of actin depolymerization in fenestrae formation. Using our fenestrae morphogenesis assay, we will determine the spatio-temporal relationship between actin disassembly and cell thinning, and will elucidate the roles of actin nucleators and actin depolymerizers. In Aim 2 we will define the mechanism of fenestrae pore formation. We will use live-cell microscopy to measure the timing of PV1 delivery to the cell surface, and identify the intracellular compartment(s) from which exocytosed PV1 is derived. In Aim 3 we will define the determinants of fenestrae diaphragm assembly. We hypothesize that PV1 is the major FD structural component, but requires additional proteins for functional FD assembly. We will use a combination of biochemistry and cellular complementation to test PV1 oligomerization structurally and functionally, and will test five PV1 interacting proteins we recently identified for roles in FD assembly. We are uniquely positioned both in terms of key and necessary expertise to complete this work. These investigations will define the assembly mechanism for an intricate and physiologically relevant cellular structure, providing valuable cell biological insight that is currently lacking.